DESCRIPTION (Investigator's Abstract): this is a new R01 proposal to study mechanisms of excitotoxicity at the cellular level. The author proposes to - 1 - use confocal microscopy as well as specific intracellular dyes to dissect the involvement of calcium, mitochondria and the production of free radicals as related to neuronal death. The fundamental hypothesis is that there is functional and temporal coupling between an NMDA receptor stimulation and cytosolic calcium accumulation, intramitochondrial accumulation, production of free radicals, mitochondrial depolarization, opening of the mitochondrial transition pore and eventual neuronal death. The author proposes four specific aims. In specific aim 1 he will use primary cultures of neurons as well as laser scanning confocal microscopy to examine and correlate the above mentioned sequence temporally. In specific aim 2 he will test the hypothesis that activation of nonNMDA receptors or voltage dependent calcium channels will have less of an effect than an NMDA receptor activation on mitochondrial calcium, free radical production and membrane potential. Specific aim 3 will determine whether inhibition of the electron transport chain will impair the ability of mitochondria to accumulate calcium as well as to cause abnormal elevation of cytosolic calcium and to facilitate opening of the permeability transition pore. Lastly the author will examine lymphoblastoid cell lines derived from patients with known mitochondrial defects to determine whether these cells lines exhibit abnormal mitochondrial calcium accumulation, membrane potential and free radical production. Furthermore he will then transfect these lymphoblasts with an NMDA receptor to determine whether they are abnormally sensitive to receptor activation as compared to lymphoblastoid cell lines from normal controls.